We have begun to apply a structural genomics approach to the systematic study of signal transducing domains this year. Analysis has been completed for three domains so far, START, GAF, and VHS.The steroidogenic acute regulatory protein (StAR) regulates acute steroidogenesis in the adrenal cortex and gonads by promoting the translocation of cholesterol to the mitochondrial inner membrane, where the first step in steroid biosynthesis is catalyzed. StAR-related lipid-transfer (START) domains occur in diverse proteins involved in lipid transport and metabolism, signal transduction, and transcriptional regulation. Mutations in the StAR gene mapping within its START domain are the underlying cause of congenital lipoid adrenal hyperplasia. Here we report the 2.2 Angstrom resolution crystal structure of the START domain of human MLN64, which reveals an a+b fold built around a U-shaped incomplete b-barrel. The interior of the protein encompasses a 26 x 12 x 11 Angstrom hydrophobic tunnel that is large enough to bind a single cholesterol molecule. The StAR and MLN64 START domains bind 1 mol cholesterol per mol protein in vitro. Based on the START domain structure and cholesterol binding stoichiometry, we propose StAR acts by shuttling cholesterol molecules one at a time through the intermembrane space of the mitochondrion.GAF domains are ubiquitous motifs present in cyclic GMP-regulated cyclic nucleotide phosphodiesterases, certain adenylyl cyclases, the bacterial transcription factor FhlA, and hundreds of other signaling and sensory proteins from all three kingdoms of life. The crystal structure of the Saccharomyces cerevisiae YKG9 protein was determined at 1.9 Angstrom resolution. The structure revealed a fold that resembles the PAS domain, another ubiquitous signaling and sensory transducer. YKG9 does not bind cyclic GMP, but the isolated first GAF domain of phosphodiesterase 5 binds with Kd = 650 nM. The cyclic GMP binding site of the phosphodiesterase GAF domain was identified by homology modeling and site-directed mutagenesis, and consists of conserved Arg, Asn, Lys, and Asp residues. The structural and binding studies taken together show that the cyclic GMP-binding GAF domains form a new class of cyclic nucleotide receptors distinct from the regulatory domains of cyclic nucleotide-regulated protein kinases and ion channels.VHS domains are found at the N-termini of select proteins involved in intracellular membrane trafficking. We have solved the crystal structure of the VHS domain of the human Tom1 (target of myb 1) protein to 1.5 Angstrom resolution. The domain consists of eight helices arranged in a superhelix. The surface of the domain has two main features: (1) a basic patch on one side due to several conserved positively charged residues on helix 3; (2) A negatively charged ridge on the opposite side, formed by residues on helix 2. We compare our structure to the recently obtained structure of tandem VHS-FYVE domains from Hrs (Mao, Y., Nickitenko, A., Duan, X., Lloyd, T. E., Wu, M. N., Bellen, H., and Quiocho, F. A. (2000) Cell 100, 447-456). Key features of the interaction surface between the FYVE and VHS domains of Hrs, involving helices 2 and 4 of the VHS domain, are conserved in the VHS domain of Tom1, even though Tom1 does not have a FYVE domain. We also compare the structures of the VHS domains of Tom1 and Hrs to the recently obtained structure of the ENTH domain of Epsin-1 (Hyman, J. , Chen, H., Di Fiore, P. P., De Camilli, P., and Brunger, A. T. (2000) J. Cell. Biol. 149, 537-546). Comparison of the two VHS domains and the ENTH domain reveal a conserved surface, composed of helices 2 and 4, that is utilized for protein-protein interactions. In addition, VHS-domain-containing proteins are often localized to membranes. We suggest that the conserved positively charged surface of helix 3 in VHS and ENTH domains play a role in membrane binding.